The green microalga Botryococcus braunii produces hydrocarbon oils at 25–75% of its dry weight and is a promising source of biofuel feedstock. Few studies have examined this species’ ecology in natural habitats, and few wild genetic resources have been collected due to difficulties caused by its low abundance in nature. This study aimed to develop a real-time PCR assay for specific detection and quantification of this alga in natural environments and to quantify spatiotemporal variations of wild B. braunii populations in a tropical pond. We designed PCR primers toward the hydrocarbon biosynthesis gene SSL-3 and examined amplification specificity and PCR efficiency with 70 wild strains newly isolated from various environments. The results demonstrated that this PCR assay specifically amplified B. braunii DNA, especially that of B-race strains, and can be widely used to detect wild B. braunii strains in temperate and tropical habitats. Field-testing in a tropical pond suggested a diurnal change in the abundance of B. braunii in surface water and found B. braunii not only in surface water, but also at 1–1.5 m deep and in bottom sediments. This method can contribute to efficient genetic resource exploitations and may also help elucidate the unknown ecology of B. braunii.

Algal biofuel research aims to make a renewable, carbon–neutral biofuel by using oil-producing microalgae. The freshwater microalga Botryococcus braunii has received much attention due to its ability to accumulate large amounts of petroleum-like hydrocarbons but suffers from slow growth. We performed a large-scale screening of fast-growing strains with 180 strains isolated from 22 ponds located in a wide geographic range from the tropics to cool-temperate. A fast-growing strain, Showa, which recorded the highest productivities of algal hydrocarbons to date, was used as a benchmark. The initial screening was performed by monitoring optical densities in glass tubes and identified 9 wild strains with faster or equivalent growth rates to Showa. The biomass-based assessments showed that biomass and hydrocarbon productivities of these strains were 12–37% and 11–88% higher than that of Showa, respectively. One strain, OIT-678 established a new record of the fastest growth rate in the race B strains with a doubling time of 1.2 days. The OIT-678 had 36% higher biomass productivity, 34% higher hydrocarbon productivity, and 20% higher biomass density than Showa at the same cultivation conditions, suggesting the potential of the new strain to break the record for the highest productivities of hydrocarbons.

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